In 2008, New England Journal of Medicine reported that one case of immunotherapy on advanced melanoma was successful, and the patient had developed multiple metastases in the body, all of which disappeared after autologous CD4+T cell treatment, with 26 months of long-term survival via a follow-up. In 2010, FDA approved autologous immune cell therapy technology of Dendreon Corporation for the clinical application of prostate cancer, and in 2011, three scientists engaged in cancer immunotherapy also was awarded Nobel Prize for medicine, suggesting a broad prospect of immunotherapy in malignant treatment.
In recent years, the use of genetically modified and induced T cell immunotherapy has achieved good results on tumor, which points out a new direction for developing immune cell therapy of tumor. This suggests a broad prospect of immunotherapy in malignant treatment.
The immune cell therapy technology has undergone the development of LAK, CIK, DC-CIK and now is rapidly growing toward antigen-loading DC-induced T-cell (DiKat), genetically modified DC-induced T cells (AV/LV-DC-CTL) and genetically modified chimeric antigen receptor T cells (CAR-T). Currently, Novartis announced a result of B lymphoma clinical trial for the CD19 target with a rate of complete efficacy of 93%, further highlighting the prospect of the immune cell therapy of cancer. However, CAR-T is challenged because of its complex cell preparation process, as well as high costs (the cost of each course is expected to be 500,000 US dollars), and currently shows a good efficacy only for CD19-positive B lymphoma, suggesting its obvious limitations.
An urgent need exists for solving the problem on how to develop an immune cell therapy technology, which has enhanced specificity and effectiveness and the advantages of simple and convenient use etc.
Contents of Invention
The present invention provides a bispecific antibody, as well as a preparation method therefor and an application thereof, and particularly a bispecific antibody capable of being combined with immune cells to enhance a targeting tumor killing capability, as well as a preparation method therefor and an application thereof.
To this end, the present invention adopts the following technical solutions:
In a first aspect, the present invention provides a bispecific antibody capable of being combined with immune cells to enhance a targeting tumor killing capability, said bispecific antibody comprising a first antibody moiety that binds to an antigen expressed on an effector T cell and a second antibody moiety that binds to an antigen expressed on a target cell.
The second antibody moiety described herein may be one or more antibodies that can bind to the target cell.
In the present invention, the first antibody moiety and the second antibody moiety are connected by a nanomaterial. The connection can be made by using the carboxyl group of the nanoparticle surface and the amino group of the antibody.
A “multispecific antibody” is an antibody that may simultaneously bind to at least two targets with different structures (e.g., two different antigens, two different epitopes on the same antigen, or a hapten and/or antigen or epitope). A “bispecific antibody” is an antibody that can bind to two targets with different structures simultaneously. The “multispecific antibody” or “bispecific antibody” described herein includes multiple or two antibodies (e.g., two monoclonal antibodies) that are connected by a nanomaterial and bind to different targets. The multispecific antibody or bispecific antibody described herein may have at least one antibody that specifically binds to a T cell and at least one antibody that specifically binds to an antigen produced by a diseased cell, tissue, organ or pathogen, or an antigen related thereto (e.g., a tumor-associated antigen), which antibodies are connected by a nanomaterial.
For the purpose of immune cell targeting therapy, the present invention utilizes the specific binding ability of an antibody to attach the antibodies capable of specifically recognizing a tumor cell and a tumor killer cell to a clinically available degradable nanomaterial, so as to form a bispecific antibody. In contrast, most anti-cancer monoclonal antibodies as a drug for the treatment of diseases are mainly based on their inherent biological functions, including complement-mediated cytotoxicity (CDC), antibody-dependent cell-mediated cytotoxicity (ADCC), apoptosis induction, opsonophagocytosis etc. The bispecific antibody of the invention not only specifically recognizes and binds to a tumor cell, but also recognizes and binds to a killer T lymphocyte, thereby assisting the T cell to recognize the tumor cell and narrowing the physical distance between the tumor cell and the killer T lymphocyte, and thus facilitating the killing of the tumor cell by the T lymphocyte. Accordingly, the specificity and effectiveness of immune cell therapy are strengthened.
The nanomaterial described herein is a biodegradable nanomaterial.
The nanomaterial described herein is generally present in the form of nanoparticle, which is selected from a conventional degradable nanomaterial, such as any of polylactic acid-glycolic acid, polylactic acid, polycaprolactone, polybutylene glycol succinate, polyaniline, polycarbonate, glycolide-lactide copolymer or glycolide-caprolactone copolymer, or a mixture thereof, preferably, but not limited to these.
The nanomaterial used herein possesses the advantages of slow releasing and good biocompatibility etc. For example, PLGA is a pharmaceutical excipient approved by FDA, which has good biocompatibility and biodegradability, no toxicity and irritation, high strength and is easily processed and molded. It is finally decomposed into hydrated carbon dioxide by enzymatically hydrolysis in vivo, and can be completely absorbed within 3-6 months after being implanted into the body.
In the present invention, the target cell is B cell, cancer cell, or pathogen cell, etc.
Preferably, the antigen expressed on the target cell is any one of carbonic anhydrase IX, alpha-fetoprotein, alpha-actinin-4, A3, A33 antibody-specific antigen, ANG2, ART-4, B7, Ba 733, BAGE, BrE3-antigen, CA125, CAMEL, CAP-1, CASP-8/m, CCCL19, CCCL21, CD1, CD1a, CD2, CD3, CD4, CD5, CD8, CD11A, CD14, CD15, CD16, CD18, CD19, CD20, CD21, CD22, CD23, CD25, CD29, CD30, CD32b, CD33, CD37, CD38, CD40, CD40L, CD45, CD46, CD52, CD54, CD55, CD59, CD64, CD66a-e, CD67, CD70, CD70L, CD74, CD79a, CD79b, CD80, CD83, CD95, CD126, CD132, CD133, CD138, CD147, CD154, CDC27, CDK-4/m, CDKN2A, CS1, CXCR4, CXCR7, CXCL12, HIF-1alpha, colon-specific antigen-p (CSAp), CEA(CEACAM5), CEACAM6, c-met, DAM, EGFR, EGFRvIII, EGP-1, EGP-2, ELF2-M, Ep-CAM, Flt-1, Flt-3, folate receptor, G250 antigen, GAGE, gp100, GPA33, GROB, HLA-DR, HM1.24, human chorionic gonadotropin (HCG) and its subunit, HER2/neu, HER3, HMGB-1, hypoxia-inducible factor (HIF-1), HSP70-2M, HST-2, Ia, IGF-1R, IFN-γ, IFN-α, IFN-β, IL-2, IL-4R, IL-6R, IL-13R, IL-15R, IL-17R, IL-18R, IL-6, IL-8, IL-12, IL-15, IL-17, IL-18, IL-23, IL-25, IL-33, insulin-like growth factor-1 (IGF-1), KC4-antigen, KS-1-antigen, KS1-4, L1CAM, Le-Y, LDR/FUT, macrophage migration inhibitory factor (MIF), MAGE, MAGE-3, MART-1, MART-2, NY-ESO-1, TRAG-3, mCRP, MCP-1, MICA, MICB, MIP-1A, MIP-1B, MIF, MUC1, MUC2, MUC3, MUC4, MUC5ac, MUC13, MUC16, MUM-1/2, MUM-3, NCA66, NCA95, NCA90, pancreatic cancer mucin, placental growth factor, p53, PLAGL2, prostate acid phosphatase, PSA, PRAME, PSMA, PlGF, ILGF, ILGF-1R, IL-6, IL-25, ROR-1, RS5, RANTES, T101, SAGE, 5100, survivin, survivin-2B, TAC, TAG-72, tenascin, TRAIL receptor, TNF-alpha, Tn antigen, Thomson-Friedrich antigen, tumor necrosis antigen, TROP-2, VEGFA, VEGFR, ED-B fibronectin, WT-1, 17-1A-antigen, complement factor C3, C3a, C3b, C5a, C5, angiogenesis marker, bc1-2, bc1-6, Kras, cMET, oncogene product, HIV virus, Mycobacterium tuberculosis, Streptococcus agalactiae, meticillin-resistant Staphylococcus aureus, Legionella pneumophila, Streptococcus pyogenes, Escherichia coli, Neisseria gonorrhoeae, Neisseria meningitidis, Pneumococcus, Cryptococcus neoformans, Histoplasma capsulatum, Hemophilis influenzae B, Treponema pallidum, Lyme disease spirochetes, Pseudomonas aeruginosa, Mycobacterium leprae, Brucella abortus, Rabies virus, Influenza virus, Cytomegalovirus, Type I herpes simplex virus, type II herpes simplex virus, human serum parvo-like virus, respiratory syncytial virus, varicella-zoster virus, hepatitis B virus, hepatitis C virus, measles virus, adenovirus, human T cell leukemia virus, Epstein-Barr virus, murine leukemia virus, mumps virus, vesicular stomatitis virus, Sindbis virus, lymphocyte choriomeningitis virus, Wart virus, Blue tongue virus, Sendai virus, Cat leukemia virus, Reovirus, Poliovirus, Simian virus 40, mouse mammary tumor virus, Dengue fever virus, rubella virus, West Nile virus, Plasmodium falciparum, Plasmodium vivax, Toxoplasma gondii, Trypanosoma rangeli, Trypanosoma cruzi, Trypanosoma rhodesiensei, Trypanosoma brucei, Schistosoma mansoni, Schistosoma japonicum, Babesia bovis, Elmeria tenella, Onchocerca volvulus, Leishmania tropica, Trichinella spiralis, Theileria parva, Taenia hydatigena, Taenia ovis, Taenia saginata, Echinococcus granulosus, Mesocestoides corti, Mycoplasma arthritidis, M. hyorhinis, M. orale, M. arginini, Acholeplasma laidlawii, M. salivarium and M. pneumoniae, preferably any one of CD19, CD20, CD22, CD30, CD33, CD38, CD123, Muc1, Muc16, HER2, HERS, EGFRvIII, VEGFA, CEA, GPA33, GP100, ANG2, L1CAM, ROR-1, CS1, MICA or MICB.
Preferably, the antigen expressed on the effector T cell is any one of ADAM17, CD2, CD3, CD4, CD5, CD6, CD8, CD11a, CD11b, CD14, CD16, CD16b, CD25, CD28, CD30, CD32a, CD40, CD40L, CD44, CD45, CD56, CD57, CD64, CD69, CD74, CD89, CD90, CD137, CD177, CEACAM6, CEACAM8, HLA-DRa chain, KIR, LSECtin or SLC44A2, preferably any one of CD2, CD3, CD4, CD5, CD6, CD8, CD25, CD28, CD30, CD40, CD40L, CD44, CD45, CD69 or CD90.
In a second aspect, the present invention also provides a method of producing a bispecific antibody as described in the first aspect of the invention comprising connecting the nanomaterial to the first antibody moiety and the second antibody moiety.
The method for producing a bispecific antibody according to the present invention comprises the steps of:
(1) preparation, collection and activation of a nanomaterial;
(2) connecting the nanomaterial obtained in step (1) with a mixture of the first antibody moiety and the second antibody moiety.
In the step (1) of the invention, the preparation of the nanomaterial comprises: dissolving the nanomaterial completely by a solvent, stirring and adding water to form a uniform emulsion.
Preferably, the nanomaterial is any one of polylactic acid-glycolic acid, polylactic acid, polycaprolactone, polybutylene glycol succinate, polyaniline, polycarbonate, glycolide-lactide copolymer or glycolide-caprolactone copolymer, or a mixture thereof.
Preferably, the solvent is any one of acetone, butanone, methanol, ethanol or isopropanol, or a mixture thereof.
Preferably, the collection of the nanomaterial comprises: collecting the prepared nanomaterial by centrifugation, and then washing the nanomaterial by resuspending in deionized water twice.
Preferably, the activation of the nanomaterial comprises: activating the nanomaterial for 0.5-5 hours by using a mixed solvent of 1-10 mg/mL 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDS) and N-hydroxysuccinimide (NHS) at room temperature.
In the step (2) of the invention, the connection comprises: collecting the activated nanomaterial by centrifugation, and then washing the nanomaterial once with the connecting reaction solution, adding a mixture of the first antibody moiety and the second antibody moiety to be connected in an equal volume into the connecting reaction solution, and then resuspending the nanomaterial with the connecting reaction solution containing the first antibody moiety and the second antibody moiety and conducting the connecting reaction for 0.5-5 hours at room temperature. After the reaction, the nanomaterial is collected by centrifugation. The nanomaterial is washed twice in Dulbecco's phosphate buffer saline (D-PBS), and then resuspended in D-PBS and stored at 4° C.
The method for producing bispecific antibodies described herein comprises the following steps:
(1) Preparation of nanomaterials: The nanomaterial is completely dissolved in acetone at a concentration of 5 to 30 mg/mL, and the solution of the nanomaterial with acetone is added to the deionized water in 1:4 v/v of acetone and deionized water with magnetic stirred at 500 to 1500 rpm/min, to form a uniform emulsion and then continue to stir until the volatilization of acetone;
(2) Collection of nanomaterials: collecting the prepared nanomaterials by centrifugation at 8000-15000 rpm/min, and then washing the nanomaterials by resuspending in deionized water twice;
(3) Activation of nanomaterials: activating the nanomaterials by using a mixed solvent of 1-10 mg/mL 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide at room temperature for 0.5-5 hours;
(4) Connection of nanomaterials with antibodies: collecting the activated nanomaterials by centrifugation, and then washing the nanomaterials once with 0.1 M D-PBS at pH=8.0, and adding a mixture of the first antibody moiety and the second antibody moiety to be connected in an equal volume into the connecting reaction solution, and then resuspending the nanomaterials with the connecting reaction solution containing the first antibody moiety and the second antibody moiety and conducting the connecting reaction for 0.5-5 hours at room temperature. After the reaction, the nanomaterials are collected by centrifugation. The nanomaterials are washed twice in D-PBS, and then resuspended in D-PBS and stored at 4° C.
In a third aspect, the present invention also provides the use of the bispecific antibody as described in the first aspect in the manufacture of a medicament for the treatment, prevention or diagnosis of a tumor. The tumor includes, but not limited to, liver cancer, non-small cell lung cancer, small cell lung cancer, adrenocortical carcinoma, acute (chronic B) lymphocytoma, myeloma, prostate cancer, breast cancer, esophageal cancer, gastric cancer, colorectal cancer, cervical cancer, kidney cancer, bladder cancer and lymphoma.
In a fourth aspect, the present invention also provides the bispecific antibody according to any one of claims 1 to 3 for use in the treatment or prevention of a tumor. The tumor includes, but not limited to, liver cancer, non-small cell lung cancer, small cell lung cancer, adrenocortical carcinoma, acute (chronic B) lymphocytoma, myeloma, prostate cancer, breast cancer, esophageal cancer, gastric cancer, colorectal cancer, cervical cancer, kidney cancer, bladder cancer and lymphoma.
In a fifth aspect, the present invention provides a method of treating a tumor comprising administering to a subject the bispecific antibody of the invention. The tumor includes, but not limited to, liver cancer, non-small cell lung cancer, small cell lung cancer, adrenocortical carcinoma, acute (chronic B) lymphocytoma, myeloma, prostate cancer, breast cancer, esophageal cancer, gastric cancer, colorectal cancer, cervical cancer, kidney cancer, bladder cancer and lymphoma.
Compared with the prior art, the invention has at least the following beneficial effects:
(1) The bispecific antibody according to present invention can be more quickly, simply and practically produced as compared to the existing bispecific antibodies which are expressed by biological methods. The bispecific antibody according to present invention can be connected directly to the monoclonal antibodies approved on the market. Because the raw materials to be used are all clinically approved on the market, the bispecific antibody prepared according to the invention can quickly enter clinical use;
(2) Compared with immune cell therapy via the genetically-modified chimeric antigen receptor T cell (CAR-T), the bispecific antibody of the present invention has disadvantages such as biodegradability, no gene recombination, low side effect, high safety, low cost, capability to combine respective specific antibodies for various tumor cells, ease of use, etc. According to present invention, a similar effect on targeting and efficiently killing cancer cells can be achieved by administrating the bispecific antibody preparation of the present invention to a subject while returning the CTL cells induced in vitro to the subject via infusion. The resulted side effects are lower than CAR-T therapy;
(3) All of the materials of the present invention can be degraded into non-toxic and harmless products in the human body and can be degraded and metabolized shortly. Accordingly, the present invention is safer than CAR-T.
Specific Mode for Carrying Out the Invention
The invention is described in details through the Examples, with reference to the accompanying drawings.